The important biological roles that peptides and proteins play as hormones, enzyme inhibitors, substrates, gene regulators, and neurotransmitters has led to the use of peptides and/or peptide mimetics as therapeutic agents. The peptide's bioactive conformation, combining structural elements such as alpha-helices, beta-sheets, turns, and/or loops, is important as it allows for selective biological recognition of receptors, enzymes, and nucleic acids, thereby influencing cell-cell communication and/or controlling vital cellular functions, such as metabolism, immune defense, and cell division (see, e.g., Babine et al., Chem. Rev. (1997) 97:1359). Unfortunately, the utility of peptides as drugs is severely limited by several factors, including their rapid degradation by proteases under physiological conditions, their poor cell permeability, and their lack of binding specificity resulting from conformational flexibility. Moreover, alpha-helical peptides have a propensity for unraveling and forming random coils, which are, in most cases, biologically less active, or even inactive, and are highly susceptible to proteolytic degradation.
Many research groups have developed strategies for the design and synthesis of more robust peptides as therapeutics. For example, one strategy has been to incorporate more robust functionalities into the peptide chain while still maintaining the peptide's unique conformation and secondary structure (see, e.g., Gante, Angew. Chem. Int. Ed. Engl. (1994) 33:1699-1720; Liskamp, Recl. Trav. Chim. Pays-Bas (1994) 113:1; Giannis, Angew. Chem. Int. Ed. Engl. (1993) 32:1244; Bailey, Peptide Chemistry, Wiley, New York (1990), 182; and references cited therein). Another approach has been to stabilize the peptide via covalent cross-links (see, e.g., Phelan et al., J. Am. Chem. Soc. (1997) 119:455; Leuc et al., Proc. Natl. Acad. Sci. USA (2003) 100: 11273; Bracken et al., J. Am. Chem. Soc. (1994) 116:6432; Yan et al., Bioorg. Med. Chem. (2004) 14:1403). However, the majority of reported approaches involved the use of polar and/or labile cross-linking groups.
“Peptide stapling” is a term coined for a synthetic methodology used to covalently join two olefin-containing side chains present in a polypeptide chain by ring closing metathesis (RCM) (see, e.g., Blackwell et al., J. Org. Chem. (2001) 66:5291-5302; Blackwell et al., Angew. Chem. Int. Ed. (1998) 37:3281). Stapling of a polypeptide using a hydrocarbon cross-linker created from an olefin metathesis reaction has been shown to help maintain a peptide's native conformation, particularly under physiological conditions (see, e.g., U.S. Pat. Nos. 7,192,713; 7,723,469; 7,786,072; U.S. Patent Application Publication Nos: 2010-0184645; 2010-0168388; 2010-0081611; 2009-0176964; 2009-0149630; 2006-0008848; PCT Application Publication Nos: WO 2010/011313; WO 2008/121767; WO 2008/095063; WO 2008/061192; WO 2005/044839; Schafmeister et al., J. Am. Chem. Soc. (2000) 122:5891-5892; Walensky et al., Science (2004) 305:1466-1470). The stapled polypeptide strategy in which an all-hydrocarbon cross-link is generated by olefin metathesis is an efficient approach to increase the helical character of polypeptides to target α-helical binding motifs. Unlike their unstapled analogues these hydrocarbon-stapled polypeptides have shown to be α-helical, protease-resistant, and cell permeable.
There are sixty known RAB GTPase isoforms and six RAB-family interacting protein (RAB-FIP or FIP) isoforms known. Each FIP isoform contains a highly conserved C-terminal RAB-binding domain (RabBD) of approximately thirty amino acids (see, e.g., Stenmark et al., Genome Biol (2011) 2:3007) important for RAB-FIP binding interactions and subsequent biological function. The conserved RabBD has a partial alpha-helical structure that undergoes a conformational change upon RAB binding. RAB function is coupled through effector proteins, including GTP transferases, GAP proteins, geranyl transferases, and RAB Coupling Proteins/RAB family interacting proteins (FIPs). Some diseases such as those resulting in bleeding and pigmentation disorders (e.g., Griscelli syndrome, Hermansky-Pudlak syndrome), mental retardation, neuropathy (e.g., Charcot-Marie-Tooth (CMT) disease), kidney disease (e.g., tuberous sclerosis), and blindness (e.g., choroideremeia) arise from direct loss of function mutations of RAB GTPases or associated regulatory molecules (see, e.g., Stein et al., Advanced Drug Delivery Reviews (2003) 55:1421-1437). In contrast, in a number of cancers (e.g., prostate, liver, breast, ovarian) as well as vascular, lung, and thyroid diseases, the overexpression of certain RAB GTPases have been correlated with disease pathogenesis (see, e.g., Stein supra; Chia et al., Biochimica et Biophysica Acta (2009) 2:110-116; Cheng et al., Nature Medicine (2004) 10:1251-1256). The development of compounds that target RAB proteins and modulate the endocytic protein trafficking pathway is a worthwhile effort in the search for new and improved therapeutics.